PROJECT SUMMARY While chimeric antigen receptor (CAR) T-cells can be very effective in advanced hematological malignancies, autologous products often have variable potency and require complex and expensive manufacturing, limiting their scalability and accessibility. The long-term goal of this proposal is to develop a well-characterized, ?off- the-shelf? (OTS) therapeutic T-cell platform using banked T-cells pre-manufactured from healthy donors, thus offering immediate availability and high potency at a reduced cost. One major limitation of this approach is potential immune rejection of infused OTS T-cells by host T- and NK-cells, which would impair persistence and clinical benefit of the T-cell therapy. Therefore, my graduate dissertation project (Aim 1) focuses on engineering OTS therapeutic T-cells to resist host immune rejection. I have developed the ?first-in-class? chimeric alloimmune defense receptor (ADR) which enables allogeneic OTS CAR T-cells to defend themselves by selectively eliminating activated host alloreactive lymphocytes while sparing other resting non-alloreactive cells. T-cells co-expressing a 4-1BB-directed ADR and a CAR evade immune rejection and produce long-term anti- tumor activity in mouse models of OTS CAR T-cell therapy for both liquid and solid tumors. We are now optimizing the 4-1BB-specific ADR for clinical translation and will initiate a Phase I clinical study in our center. I am also exploring other potential ADR targets, including OX40 and CD40L, to maximize the anti-rejection activity. In addition to alloimmune rejection, activity of OTS T-cells in solid tumors can be inhibited by the immunosuppressive tumor microenvironment (TME). Mounting evidence suggests that the inflammatory milieu created by therapeutic T-cells may elicit reactive changes both locally (in the TME) and systemically (in circulation) that further inhibit anti-tumor activity of therapeutic T-cells and possibly promote tumor growth and metastasis. Examples include a surge of immunosuppressive M2-like macrophages in neuroblastoma patients receiving GD2 CAR T-cells and poor responses to CD19 CAR T-cell therapy in patients with high circulating myeloid-derived suppressor cells. In addition, preclinical studies indicate that treatment-induced inflammation enhances pre-metastatic niche (PMN) formation and increases the risk of metastasis. Therefore, during my post- doctoral training (Aim 2), I will first elucidate the reactive changes (both in TME and in circulation) caused by therapeutic T-cells and identify cellular/molecular mediators of enhanced immunosuppression at the primary tumor site. I will also investigate how T-cell therapies may affect PMN formation in solid tumors. I will then further modify therapeutic T-cells to counteract these unwanted responses by arming them with secreted factors (antibodies, peptide inhibitors) to block the responsible cytokines / chemokines, or by enabling them to selectively eliminate inhibitory cellular subsets in the TME. Successful completion of both Aims will ultimately improve the efficacy of OTS T-cell therapies of cancer.